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Wiskott-Aldrich Syndrome/X-Linked Thrombocytopenia: WASP Gene
Mutations, Protein Expression, and Phenotype
By Qili Zhu, Chiaki Watanabe, Ting Liu, Diane Hollenbaugh, R. Michael Blaese, Steven B. Kanner, Alejandro Aruffo,
and Hans D. Ochs
Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), caused by mutations of the WAS protein
(WASP) gene, represent different phenotypes of the same
disease. To demonstrate a phenotype/genotype correlation,
we determined WASP gene mutations in 48 unrelated WAS
families. Mutations included missense (20 families) and nonsense (eight) mutations located mostly in exons 1 to 4, and
splice-site mutations (seven) and deletions and insertions
(13) located preferentially in exons 7 to 11. Both genomic
DNA and cDNA were sequenced and WASP expression was
measured in cell lysates using peptide-specific rabbit antiWASP antibodies. WASP was expressed in hematopoietic
cell lines including bone marrow–derived CD34" cells. Missense mutations located in exons 1 to 3 caused mild disease
in all but one family and permitted WASP expression, although frequently at decreased concentration. Missense
mutations affecting exon 4 were associated with classic
WAS and, with one exception, barely detectable WASP. Nonsense mutations caused classic WAS and lack of protein.
Insertions, deletions, and splice-site mutations resulted in
classic WAS and absent, unstable, truncated, or multiply
spliced protein. Using affinity precipitation, WASP was
found to bind to Src SH3-containing proteins Fyn, Lck, PLCg, and Grb2, and mutated WASP, if expressed, was able to
bind to Fyn-glutathione S-transferase (GST) fusion protein.
We conclude that missense mutations affecting the PH domain (exons 1 to 3) of WASP inhibit less important functions
of the protein and result in a mild phenotype, and that missense mutations affecting exon 4 and complex mutations
affecting the 3* portion of WASP interfere with crucial functions of the protein and cause classic WAS.
q 1997 by The American Society of Hematology.
W
to polysaccharides.3,4 Antibody responses to certain T-cell–
dependent antigens such as bacteriophage fX174 are quantitatively reduced with lack of amplification and failure to
switch from IgM to IgG.4 Abnormal function of WAS T
cells is suggested by diminished but not absent lymphocyte
responses to mitogens,3 depressed proliferative responses to
allogenic cells4 and immobilized anti-CD3 monoclonal antibody,6 and failure to proliferate in response to periodate.7
Distinguishable from the classic WAS phenotype is a milder
form designated as hereditary X-linked thrombocytopenia
(XLT).7-11 In patients with XLT, eczema is mild, if present,
and immune functions may be normal. The genes for both
WAS and XLT have been mapped to the pericentromeric
short arm of the X chromosome at Xp 11.22,12,13 and sequence analysis of the WASP gene has identified mutations
of the same gene in both phenotypes.14,15
The gene responsible for WAS is composed of 12 exons
containing 1,823 base pairs and encodes a 502–amino acid
protein14 that appears to be of central importance for the
function of hematopoietic cells. WASP, expressed in all hematopoietic stem cell–derived lineages,14 is located predominantly in the cytoplasm.16-19 Although several unique binding domains of WASP have been identified, its precise
function is unknown. WASP has a proline-rich region with
motifs corresponding to the PXXP binding consensus for
SH3 domains.20 Interaction of WASP with SH3 domains of
selected signaling molecules has been demonstrated, including p47nck, a 47-kD cytosolic adapter protein,16 Fyn,21
cFRG,21,22 c-Src, p47phox,22 Grb2,23 and the Tec family cytoplasmic tyrosine kinases, Btk, Tec, PLC-g1,22,23 and Itk.23,24
Of these, only p47nck and Fyn were shown to coprecipitate
in vivo with WASP. In addition, WASP may play a role in
the regulation of the actin/cytoskeleton system by interacting
directly with the Rho-like GTPase, cdc42.25-27
To investigate a possible correlation between genotype
and phenotype, we identified the mutations of the WASP
gene in a large cohort of patients with WAS or XLT, determined the effect of these defects on gene transcription, protein expression, and SH3 binding, and correlated the molecular findings with the clinical phenotype.
ISKOTT-ALDRICH SYNDROME (WAS), an Xlinked recessive disorder with variable clinical phenotype,1,2 is caused by mutations of the WAS protein
(WASP) gene. Thrombocytopenia and small platelets are
characteristic findings present in all WAS patients irrespective of clinical severity. Patients with a classic WAS phenotype present with eczema that may be severe, develop recurrent bacterial and viral infections due to abnormal immune
function, and have an increased risk of autoimmune diseases
and malignancies.1-5 Immunologic abnormalities typically
observed in WAS are complex and involve both B- and Tcell function. Affected male infants have a normal number
of circulating lymphocytes but develop lymphopenia by 6
to 8 years of age due to a loss of T lymphocytes.4 Most
affected boys present with normal levels of serum IgG, moderately depressed IgM, and elevated IgA and IgE. Antibody
responses are normal to some antigens and insufficient to
others. A consistent finding is a markedly depressed response
From the Division of Infectious Diseases, Immunology and Rheumatology, Department of Pediatrics, University of Washington
School of Medicine, Seattle; Bristol-Myers Squibb Pharmaceutical
Research Institute, Seattle, WA; and the Clinical Gene Therapy
Branch, National Center for Human Genome Research, National
Institutes of Health, Bethesda, MD.
Submitted December 19, 1996; accepted May 27, 1997.
Supported by grants from the National Institutes of Health
(HD17427), the March of Dimes Birth Defects Foundation (FY960330), and the DeJoria Wiskott-Aldrich Research Fund; conducted
in part at the Clinical Research Center at the University of Washington (RR-37).
Address reprint requests to Hans D. Ochs, MD, Division of Infectious Diseases, Immunology and Rheumatology, Department of Pediatrics, University of Washington School of Medicine, Box 356320,
Seattle, WA 98195-6320.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/9007-0015$3.00/0
Blood, Vol 90, No 7 (October 1), 1997: pp 2680-2689
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WAS PROTEIN EXPRESSION AND PHENOTYPE
2681
MATERIALS AND METHODS
Patients and clinical phenotypes. Affected members of 48 unrelated WAS families were included in this analysis. Using previously
published criteria,19,28 the severity of WAS-associated symptoms was
estimated and expressed as a score of 1 to 5. A score of 1 was
assigned to a patient with thrombocytopenia and small platelets without any other symptoms, clinical findings, or laboratory abnormalities. Patients with platelet abnormalities and mild, transient eczema
with or without minor infections received a score of 2. Patients with
persistent but manageable eczema or recurrent infections or both
received a score of 3. Patients with persistent and difficult-to-control
eczema and frequent life-threatening infections were scored as a 4.
A score of 5 was assigned if patients presenting with eczema and/or
frequent infections developed autoimmune diseases or malignancies.
Cell lines. B-lymphoblastoid cell lines (B-LCLs) were established by inoculating peripheral blood mononuclear cells (PBMCs)
from normal controls and WAS patients with Epstein-Barr virus
(EBV)-containing supernatant, as previously described.29 The human
kidney tumor cell line 293 and the hepatoma cell line SK HEP1
(both provided by Dr Mark Kay, University of Washington, Seattle,
WA), the human megakaryoblastic cell line MEG-01 (a gift from
Dr Thalia Papayannopoulou, University of Washington, Seattle), and
HEL, HE-60, Jurkat T-cell line, and K562 cells (obtained from the
American Type Culture Collection, Rockville, MD) were maintained
in RPMI 1640 supplemented with 10% fetal bovine serum. CD34/
bone marrow cells from a normal human donor were prepared by
Dr Robert Andrews (University of Washington, Seattle).
RNA isolation and reverse transcriptase–polymerase chain reaction. RNA isolation, using a single-step method and Trizol
(GIBCO-BRL, Gaithersburg, MD), and reverse transcriptase–polymerase chain reaction (RT-PCR), using the SuperScript Preamplification System kit (GIBCO-BRL), were performed as previously described.30 WASP cDNA was amplified by PCR in two overlapping
fragments using the previously reported primers28 and the Expand
High Fidelity PCR System (GIBCO-BRL). PCR products were purified by agarose gel electrophoresis.
Dideoxynucleotide fingerprinting and direct sequencing of cDNA.
To screen the amplified WASP cDNA for mutations, we modified
the dideoxynucleotide fingerprinting (ddF) method originally designed by Sarkar et al31 as previously described,28 using the fmol
cycle-sequencing kit (Promega, Madison, WI). The resulting PCR
products were electrophoresed in 5% polyacrylamide nondenaturing
gels at 47C. The mutations suggested by ddF were confirmed by
direct sequencing using selected primers and the fmol cycle-sequencing kit.
DNA purification and sequencing of genomic DNA. DNA was
extracted from B-LCLs as previously described.30 Purified genomic
DNA samples were amplified with primer pairs designed to span
the suspected mutation sites, including exon/intron junctions, using
previously reported conditions.28 The amplified DNA fragments were
separated by agarose gel electrophoresis and directly sequenced using the fmol cycle-sequencing kit.
Northern blot analysis. PolyA(/) mRNA samples isolated from
30 mg total RNA with the polyATtract mRNA isolation system
(Promega) were electrophoresed through agarose/formaldehyde gels
and transferred to nylon membranes (Magna NT; MSI, Westboro,
MA). The WASP cDNA clone M5.5, a 750–base pair cDNA fragment of the WAS gene, was radiolabeled by random primer extension and used as a probe for hybridization. A human actin cDNA
probe was used to monitor mRNA loading.
Anti-WASP antisera. Three hydrophobic peptide sequences were
selected from the reported WASP sequence.14 Peptide 1050 (ADEDEAQAFRALVQEK) represents part of exon 4, peptide 1048 (SSRYRGLPAPGPSPADKK) part of exon 7, and peptide 1049 (KRS-
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RAIHSSDEGRDQ) part of exons 11 and 12. Following synthesis,
the peptides were coupled to ovalbumin, suspended in Freund adjuvant, and used to immunize rabbits to raise polyclonal anti-WASP
antisera. The antisera were designated Ab953 (peptide 1050), Ab503
(peptide 1048), and Ab1468 (peptide 1049). Specificity was determined by Western blot analysis of in vitro translated WASP gene
product and lysates from normal B-LCLs.
Western blot analysis. B-LCLs from normal control subjects
and WAS patients were suspended at 2.5 1 107/mL in lysis buffer
containing 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mmol/
L PMSF, 0.5% aprotinin, and 10 mg/mL leupeptin at pH 7.5 and
kept on ice for 10 minutes. The protein concentration was determined
in each lysate by a protein assay kit (Bio-Rad Laboratories, Hercules,
CA). From each sample, 40 mg total protein was loaded onto a
sodium dodecyl sulfate (SDS)-polyacrylamide gel, electrophoresed,
and transferred to a PVDF Immobilon-P membrane (Millipore, Bedford, MA). After blocking with 10% nonfat milk, the membranes
were incubated with anti-WASP antibody 503 (and, if indicated,
with antibodies 953 or 1468) at 1:5,000 dilution for 1 hour at room
temperature. After washing with 0.1% Tween/phosphate-buffered
saline four times, the membranes were incubated with horseradish
peroxidase–conjugated goat anti-rabbit Igs (Biosource International,
Camarillo, CA) at a concentration of 1:10,000 for 1 hour at room
temperature. The results were visualized by an enhanced chemiluminescence method (Amersham, Arlington Heights, IL).
Affinity precipitation of WASP binding proteins. B-LCL lysates
containing 80 mg total protein were incubated with 1 mg SH3 (Fyn,
Lck, GAP, PLC-g, and Grb2) or SH2 (Fyn, GAP, PI-3, and PLCg) glutathione S-transferase (GST) fusion protein agarose beads
(UBI, Lake Placid, NY) and 25 mL Sepharose in 500 mL lysis buffer
at 47C for 2 hours. After extensive washing, the pelleted beads were
incubated with 20 mL 21 loading buffer at 957C for 3 minutes,
and the supernatants were loaded onto SDS-polyacrylamide gel for
electrophoresis. The membranes were incubated with anti-WASP
antibody 503.
In vitro translation of WASP. The entire coding sequence of
WASP cDNA was amplified using the Expand High Fidelity PCR
System (GIBCO-BRL) and primers with restriction enzyme sites (5*AAG ACA GGA TCC AGA AAG CAC CAT GAG TGG, forward
[BamHI, cDNA position 25] and 5*-GGC CFA TCT AGA CTC
AGC CAC TCA GTC ATC, reverse [Xba I, cDNA position 1554]).
The digested PCR products and the pd16 vector were ligated using
T4 DNA ligase (GIBCO-BRL) and competent cells (DH5a) transformed using standard electroporation. Using the TNT in vitro Translation system (Promega) and a nonradiolabeled amino acid mixture,
plasmid DNA was translated and the product was used for Western
blot analysis.
RESULTS
WASP-specific antisera. Expression of WASP was determined by rabbit anti-WASP antibodies raised against peptides encoded by exon 4 (Ab953), exon 7 (Ab503), and
exon 11 (Ab1468) (Fig 1A). Postimmunization sera but not
preimmunization sera recognized a band of 62 kD in Western
blots generated from normal B-LCL lysates (Fig 1B). Ab503
had the highest titer and the least background, and was used
in most experiments. Figure 1C shows that the protein translated from the pd16-WASP plasmid DNA, but not that expressed by a control plasmid containing the luciferase gene,
was recognized by the anti-WASP Ab503, demonstrating
that the antibody was specific for WASP.
WASP is expressed by hematopoietic cells. Previous
studies demonstrated that WASP mRNA was expressed in
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2682
ZHU ET AL
Fig 1. Anti-WASP antisera. (A) WASP-derived peptides used in the production of rabbit anti-WASP antibody (Ab). (B) Assessment of Ab953,
Ab503, and Ab1468 using Western blot analysis of normal B-LCL lysate and preimmunization and postimmunization antisera. (C) Only protein
translated from WASP gene mRNA, not from luciferase gene mRNA, was recognized by Ab503 as a single band of 62 kD, demonstrating that
the antibody was specific for WASP.
all hematopoietic cells.14 To examine WASP expression at
the protein level, we studied cell lysates from unseparated
and CD34/-enriched human bone marrow cells and several
human cell lines by Western blot analysis. WASP was abundant in lysates of MEG-01, HEL, HL-60, and cultured normal T- and B-cell lines, and was detectable in lysates of
bone marrow, CD34/ cells, and Jurkat and K562 cells (Fig
2). WASP was not detected in the human kidney tumor cell
line 293, the hepatoma cell line SK Hep 1, or a B-LCL
derived from a WAS patient with a nonsense mutation in
exon 1 (DM, Table 1).
Mutation analysis. RT-PCR products obtained from
PBMCs or B-LCLs derived from patients with WAS or XLT
were screened by the ddF method. Mutations were confirmed
by direct sequencing of the RT-PCR products and of amplified genomic DNA. Patients with multiple RT-PCR products
and/or splice-site mutations were further studied by subcloning the cDNA and sequencing up to 20 individual clones.
Mutations of the WASP gene were identified in 59 affected
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members of 48 unrelated families. Table 1 summarizes the
results, dividing patients into groups with mild (scores 1 to
2) and classic (scores 3 to 5) WAS phenotypes. Mutations
in 17 of these families were previously reported by us14,28,34
or by others (Table 1).32,33 Of 48 mutations identified, 40 are
unique within this patient population, and compared with
the mutations reported to date,14,15,28,32-40 20 are novel and
found only in our patient population. The mutations identified are distributed throughout the WASP gene and affect
all exons except 5, 8, and 12. All but one of 20 missense
mutations identified are located in exons 1 to 4. Eight unrelated families had five unique nonsense mutations located in
exons 1, 3, 7, 9, and 10, including patient RM, in whom the
nonsense mutation affects codon 99 in exon 3, resulting in
two transcripts, one containing the stop codon and the other
deleting exon 3. Five of the six unique point mutations (from
seven families) resulting in abnormal splicing were located
in exons/introns 6 to 11. Deletions and insertions of genomic
DNA were identified in 13 unrelated patients, representing
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WAS PROTEIN EXPRESSION AND PHENOTYPE
2683
Fig 2. Expression of WASP in human cell lines. Western blot analysis of lysates from various hematopoietic cell lines (HL-60, K562, HEL,
MEG01, Jurkat, normal IL-2–dependent T-cell line, normal B-LCL, and
a B-LCL from a WAS patient with the classic phenotype) and 2 nonhematopoietic cell lines (293 and SK HEP1). All hematopoietic cell lines
express WASP, with the exception of the B-LCL derived from a WAS
patient. Nonhematopoietic cell lines do not express WASP.
12 unique mutations, and were located either in exons 1 to
3 (six patients) or exons 10 and 11 (seven patients). Mutation hotspots observed include the known hotspots C290T
(Arg86Cys), G291A (Arg86His), and C665T (Arg211 r
stop) and the two splice-site mutations, t r c / 2 on intron
9 and t r c / 2 on intron 11 (X2). In addition, a series of
five guanines at positions 1301 to 1305 were involved in
three unrelated families: a G insertion resulting in a
frameshift and stop at amino acid 494 was observed in two
families, and a G deletion resulting in a frameshift and stop
at amino acid 444 was observed in the third family.
WASP expression by cells from WAS/XLT patients. BLCLs from affected males of 41 unrelated families and frozen
PBMCs from two additional unrelated males were available
for Western blot analysis of WASP (Table 1). Selected blots
are shown in Fig 3. As expected, all eight patients with nonsense mutations, including three patients (TH, Kpe, and AT)
not shown in the figure, had a classic phenotype with scores
of 3 to 5 and failed to express WASP (Table 1 and Fig 3A).
Five of 20 unrelated patients with missense mutations had
transitional mutations in exon 4. All five presented with a
severe phenotype and had either WASP that was normal in
amount and size (CC) or WASP that was barely detectable
by Western blot and appeared slightly shorter (MS, CH, and
ST) (Table 1 and Fig 3A), although gene expression measured
by Northern blot analysis was normal in the three patients
studied (Table 1). Of the remaining 15 unrelated patients with
missense mutations, 14 had mutations located in exons 1 to
3 and one in exon 9; all but one had a mild phenotype,
and where determined, all had normal mRNA expression and
detectable WASP by Western blot analysis (Table 1 and Fig
3A). WASP expression was either markedly decreased as in
patient TS (9% of normal by densitometry) or near normal
as in patients MM (75%) or JH (60%).
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Seven unrelated patients had point mutations affecting
either a regular or cryptic splice site or a nonsense mutation
resulting in exon-skipping (RM). In patient BE with XLT
and a score of 1, we found two splicing products: 70%
of 20 RT-PCR clones generated from a B-LCL showed an
insertion of 38 nucleotides derived from intron 6 resulting
in frameshift and premature stop; however, 30% of the
clones had a normal cDNA sequence and, on Northern blot,
a normal quantity of mRNA. The presence of normal-size
WASP in a cell extract, although considerably decreased in
quantity, confirms this explanation (Table 1 and Fig 3B).
The other six patients with splice-site mutations had classic
WAS phenotypes with scores of 3 to 5 and either a complete
absence of WASP (SD, DMa, MB, and RM) or the presence
of truncated WASP (BM and KP). The latter two patients,
who are unrelated, have identical mutations that result in
deletion of exon 11, frameshift, and loss of the termination
codon (TGA). The pattern of multiple bands observed in
Western blots suggests that the use of normally nontranslated
sequences of exon 12 render WASP unstable.
The remaining 13 patients had deletions or insertions of
one or more nucleotides affecting genomic DNA. All but
one (JVT) had classic WAS and a score of 3 to 5. WASP,
evaluated in 11 patients, was either truncated or unstable or,
as in patient RP, not demonstrable (Fig 3B). Patients RW
and AG, who are unrelated but have the same mutation,
showed multiple bands of WASP, suggesting unstable protein or, less likely, multiple splicing products.
In vitro interaction of WASP and SH3 domains. To determine whether wild-type WASP interacts in vitro with proteins
containing SH3 domains, we used affinity precipitation and
Western blot analysis to assess five SH3-GST fusion proteins
(Fyn, Lck, GAP, PLC-g, and Grb2) and, as controls to rule out
nonspecific binding, seven SH2-GST fusion proteins bound to
agarose beads. If the beads were incubated with normal BLCL lysate and the eluted proteins subjected to Western blot
analysis, SH3 domains of Fyn, Lck, PLC-g, and Grb2 were
found to bind to WASP in vitro (Fig 4A). However, the SH3
domain of GAP and all SH2 domains studied (Fyn, GAP, PI3, and PLC-g) failed to bind to WASP, suggesting that WASP
selectively binds to many SH3 proteins and that the binding
is not due to a nonspecific association. Attempts to confirm
the interactions between WASP and Fyn, Lck, PLC-g, and
Grb2 by in vivo coprecipitation experiments have been unsuccessful. To determine if mutated WASP from selected patients, known to express protein, can bind SH3-containing
protein in vivo, we used a Fyn-GST fusion protein and affinity
precipitations. Mutated WASP was precipitated in vitro by
Fyn SH3-GST fusion protein agarose beads from lysates of
B-LCLs established from 12 WAS patients. Only patients
with demonstrable WASP in the cell lysates showed a band,
as demonstrated by direct Western blot analysis (Fig 4B).
The amount of WASP precipitated by Fyn SH3-GST agarose
beads seemed to correlate with the amount of WASP in the
cell lysate (Fig 3).
DISCUSSION
The recent cloning of the WASP gene has made possible
the genetic definition, carrier detection, and prenatal diagno-
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2684
ZHU ET AL
Table 1. Patient Characteristics
Patient Initials
Score
cDNA Mutations
Mild phenotype
(XLT)
JD§
JN
JDe/JTDe
JG/AVC
Cro
JS#
TS§
MM
DS/DT/JW
JH/CSØ
AR#
JR
SJ
RC/BGØ
BE
2
2
2/2
2/2
2
2
2
1
2/2/2
2/2
1
2
2
2/2
1
C113T (Leu27Phe)
T150C (Leu39Pro)
T150C (Leu39Pro)
C168T (Thr45Met)
C168T (Thr45Met)
C174A (Ala47Asp)
C177T (Thr48Ile)
C201T (Ala56Val)
G257A (Val75Met)
C290T (Arg86Cys)
C290T (Arg86Cys)
G291A (Arg86His)
A354G (Tyr107Cys)
A953G (Met307Val)
70% ins 38 nt intron 6
(frameshift stop aa 190);
30% normal
50% del 966-1121 (inframe del
52 aa); 25% del exon 10
(frameshift, stop aa 334);
25% 1029 ins T
(Asn335stop)
JVT
2
Severe phenotype
(classic WAS)
JGo
MS#
GC
CH/ZR§
5
5
4
5/4
ST
3
CC
DM#
Kpe
AT
CMcT
MW
TH/DH#
SB/HB
RM\#
4
5
4
4
4
5
3/2†
5/5
5
EA#
3
DB
RP/EM§
4
5/5
TF
5
LL
4
SH
4
CMc
3
JSc
5
DJ§
4
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Exon
1
1
1
2
2
2
2
2
2
2
2
2
3
9
Intron 6
10
Genomic DNA
Mutations
Northern Blot
C113T
T150C
T150C
C168T
C168T
C174A
C177T
C201T
G257A
C290T
C290T
G291A
A354G
A953G
g r a, /5, intron 6
//
//
//
//
//
//
//
/
////////
/////
ND
//
//
(/)
/
1029 ins T
(/)
T138A, C139T (Leu35His)
1
T138A, C139T
G407A (Gly125Arg)
4
G407A
T417C (Phe128Ser)
4
T417C
G425A (Glu131Lys)/C290T
4
G425A
(Arg86Cys)
G425A (Glu131Lys), G431A
4
G425A, G431A
(Glu133Lys)
C435T (Ala34Val)
4
C435T
C71T (Arg13stop)
1
C71T
C71T (Arg13stop)
1
C71T
C665T (Arg211stop)
7
C665T
C665T (Arg211stop)
7
C665T
C923T (Gln297stop)
9
C923T
C995T (Arg321stop)
10
C995T
C995T (Arg321stop)
10
C995T
C329T(Gln99stop), del exon 3
3
C329T
(del 29 aa)
62-64 ins C (frameshift, stop
1
C62-64 ins
aa 37)
140-142 del TT(Phe36stop)
1
140-142 del TT
211 del T (frameshift, stop aa
2
211 del T
75)
206-210 del C (frameshift stop
2
206-210 del C
aa 75)
ND (expected: frameshift,
3
312 del G, 313
stop aa 126)
del T
1030-1035 del G (frameshift,
10
1030-1035 del G
stop aa 444)
1025 ins A (frameshift, stop
10
1025 ins A
335)
1109-1113 del C (frameshift,
10
1109-1113 del C
stop aa 444)
C1109A (Pro359Thr) / 111010
C1109A, 11101113 del C (frameshift, stop
1113 del C
aa 444)
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ND
//
ND
/////
Western Blot
(% of control)*
/ (19)
/ (12)
(3.6)
/ (46)
/ (11)
/ (9-30)
/ (9-30)
// (75)
/ (24-26)
// (34-60)
ND
/ (17)
/ (14)
// (66)
/ (3.5)
/ (truncated)
/ (30)*
(/) (õ1) (truncated?)
ND
(/) (4-10) (truncated?)
//
(/) (0.5) (truncated?)
ND
//
//
—
ND
(/)
(/)
(/)
(/)
/ (65)
—
—
—
ND
—
—
—
—
—
/
//
////
(/)*
ND
ND
ND
ND
(/)
/
—
—
/ (truncated)
(/)
(/) (truncated)
(/)
/ (truncated)
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WAS PROTEIN EXPRESSION AND PHENOTYPE
2685
Table 1. Patient Characteristics (Cont’d)
Patient Initials
Score
RW
4
AG
5
CR#
5
PP#
4
SD
3
Dma
4
MBØ#/TD‡
4/2‡
BM#
4
KP
5
cDNA Mutations
1301-1305 ins G (frameshift,
stop aa 494)
1301-1305 ins G (frameshift,
stop aa 494)
1301-1305 del G (frameshift,
stop aa 444)
1372 ins 13 nt (frameshift,
stop aa 498)
ins intron 3 (frameshift, stop
200)
del 705-768 (frameshift, stop
aa 239)
80% ins 114 nt of intron 9
(frameshift, stop aa 326);
20% ins intron 9
(frameshift, stop aa 326)
del exon 11 (1373-1487)
(frameshift, stop aa 543)
del exon 11 (1373-1487)
(frameshift, stop aa 543)
Genomic DNA
Mutations
Exon
Northern Blot
Western Blot
(% of control)*
10
1301-1305 ins G
//
// (truncated, unstable)
10
1301-1305 ins G
//
// (truncated, unstable)
10
1301-1305 del G
(/)
// (truncated, unstable)
del 9 nt, ins 11 nt,
intron 10
g r a, 01, intron 3
//
/ (truncated)
(/)
—
A705G (ATA r
gta)
t r c, /2, intron 9
—
—
// (longer)
—
10/11
Intron 3
7
Intron 9
Intron 11
Intron 11
t r c, /2, intron
11
t r g, /2, intron
11
//
/ (unstable)
/
/ (unstable)
Abbreviations: aa, amino acid; del, deletion; ins, insertion.
* Cell extracts from EBV-induced B-LCLs established from patients, except for IGo and DB, for whom only frozen PBMCs were available.
† DH, the younger brother of TH, underwent bone marrow transplantation (BMT) during his first year of life; he had platelet abnormalities
and mild eczema. Two of their uncles died in childhood of WAS, and 1 uncle is alive at 35 years of age with a score of 2.
‡ TD had a BMT at õ1 year of age, when he had platelet abnormalities and mild eczema.
§ Reported by Derry et al, 199534: JD Å DV-1, TS Å AS-1, CH/ZR Å HR 1.1.1/HR 1.1.2, RP/EM Å PM 1.1.1/PM 2.1.1, DJ Å JN 1.1.1.
\ Reported by Kwan et al, 199532: RM Å 1120.
Ø Reported by Kwan et al, 199533: CS Å 26-1038, BG Å 25-1039, MB Å 11-356.
# Reported by Zhu et al.28
sis of WAS. Mutation analysis has demonstrated that a
milder WAS phenotype described variably as XLT, ‘‘isolated’’ thrombocytopenia, and ‘‘atypical’’ or ‘‘attenuated’’
WAS is caused by mutations of the same gene responsible
for ‘‘classic,’’ ‘‘full-blown’’ WAS.14,15
Attempts to correlate these strikingly different WAS phenotypes with specific genotypes have resulted in controversial interpretations. One group of investigators reported families whose affected males presented with the same clinical
phenotypes,2,32,37 whereas others have described families
whose affected members had different phenotypes.5,38 To
assess the clinical phenotype objectively, we designed a simple scoring system based on characteristic clinical symptoms. This allowed us to differentiate patients with symptoms limited to platelet abnormalities and bleeding (XLT)
from those with a classic WAS phenotype. To define in more
detail the nature of each mutation, we not only sequenced
genomic DNA but also examined the effect of the mutations
on RNA transcription, expression of WASP in patient lymphocytes, and binding of mutated WASP to the SH3-containing protein-tyrosine kinase Fyn.
To quantify WASP, we generated rabbit polyclonal antibodies to three different peptides derived from WASP. The
antibody preparation (Ab503) we selected for most experiments was highly specific and suitable for immunostaining
and Western blot analysis. This antibody detected WASP in
all hematopoietic cell lines studied, including T- and B-cell
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lines, erythroleukemia and myelogenous leukemia cell lines,
and a megakaryocytic cell line. WASP was also present
in human bone marrow–derived CD34/ cells, but not in
nonhematopoietic cell lines. A protein, N-WASP, resembling the structure of WASP and having many of the same
functional domains has been recently identified in nonhematopoietic cells.41
To examine the effect of individual WASP mutations on
the clinical phenotype, we identified mutations of the WASP
gene in 48 unrelated families. Twenty (50%) of 40 unique
mutations observed in this group of patients have not been
reported by others.40 This heterogeneity has been observed
in other X-linked immunodeficiency disorders, and may reflect the high rate of new mutations characteristic for Xlinked disorders. The most frequent mutations identified (20
of 48) were missense mutations, all but one within exons 1
to 4. The remaining 28 mutations consisted of nonsense
mutations (eight families), deletions and insertions (13 families), and splice-site mutations (seven families) and were
located preferentially in exons 7 to 11. Deletions and/or
insertions affecting genomic DNA resulted in direct stop
codons, frameshift and early termination, or, as in patient
JVT, multiple gene products. Mutations affecting splice sites
resulted frequently in multiple splicing products.
To determine the effects of the mutations on WASP expression, we established B-LCLs from affected members of
the majority of our WAS families. Patients with missense
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2686
ZHU ET AL
Fig 3. Western blot analysis of cell lysates (B-LCLs) from WAS patients (see Table 1 for detailed description of the mutations). (A) Blots
from patients with nonsense mutations or missense mutations affecting exons 1 to 4. (B) Blots from patients with splice-site mutations,
insertions, or deletions located in exons 7 to 11 (except patient SD, who has a splice-site mutation affecting intron 3).
mutations located in exons 1 to 3, with one exception, had
mild disease (a score of 1 to 2), and cell extracts contained
WASP at concentrations that varied between 3.6% and 75%
of normal control values. The exception was JG, with a score
of 5, who had two consecutive point mutations resulting in
substitution of leucine by histidine at codon 35 and whose
PBMCs had WASP of normal size and moderately reduced
amount (30% of normal). Five patients with missense mutations affecting exon 4 presented with a classic WAS phenotype and a score of 3 to 5 and, with one exception, had
barely detectable WASP of slightly smaller size. As expected, patients with nonsense mutations lacked WASP and
were found to have a classic WAS phenotype with a score
of 4 or 5. The 5* region of WASP (exons 1 to 3) where most
missense mutations are located has been designated WH127
and has the characteristics of a PH domain.41 We hypothesize
that the amino acid substitutions (except Leu35His) located
in exons 1 to 3 of the WASP gene only partially inhibit the
function of the protein, and affect platelet number and platelet size, possibly by interfering with platelet release from
megakaryocytes.4 In contrast, exon 4 appears to be critical
for the stability and function of WASP, since most of the
missense mutations we observed in exon 4 resulted in lack of
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detectable WASP expression or in truncated/unstable protein
although normal amounts of transcript were present. The
importance of exon 4 is further underlined by the observation
that all of our patients with missense mutations in exon 4
have developed a classic severe WAS phenotype with a score
of 4 to 5. Insertions, deletions, and splice-site mutations led
to the absence of WASP, unstable or truncated WASP, or
multiple splicing products and, as a rule, were associated
with severe, classic WAS. The exceptions were two patients
with complex mutations. Further analysis provided an explanation for their mild XLT phenotype. Patient BE, with a
score of 1, has a g r a transition at nucleotide /5 of intron
6, which does not completely inactivate the splice donor site.
As a consequence, a portion of the gene transcript is normally
spliced and translated into a product of normal size by Western blot analysis. However, the dominant splicing product
of this patient uses a cryptic splice site, and contains the 5*
portion of intron 9, leading to frameshift and early termination. The other exception is patient JVT who, by genomic
DNA analysis, has a T insertion at position 1,029. This mutation is expected to result in frameshift and premature
termination three codons downstream of the T insertion
(Asn335stop). However, cloning of the RT-PCR product re-
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WAS PROTEIN EXPRESSION AND PHENOTYPE
2687
Fig 4. In vitro interaction of WASP and SH3-containing proteins. (A) Proteins containing SH2 and
SH3 domains were used to affinity-precipitate
WASP. Of 5 SH3-GST fusion proteins, 4 (Fyn, Lck,
PLC-g, and Grb2) were able to bind WASP. None of
the SH2-GST fusion proteins were able to bind
WASP. (B) Lysates of B-LCLs established from 12
WAS patients, some expressing WASP, were incubated in vitro by Fyn SH3-GST fusion protein agarose
beads. Only patients with demonstrable WASP
showed a band (JS, JH, JG, JD, TS, and possibly CH).
vealed that only approximately 25% (five clones) of WASP
cDNA derived from B-LCLs in patient JVT shows the T
insertion. Two additional cDNA species were identified, one
with a deletion of the entire 407 nucleotides of exon 10,
resulting in truncation, frameshift, and loss of the last nine
amino acids of exon 12. The third and most frequent (10 of
20 clones) cDNA species identified in patient JVT had an
inframe deletion of the first 52 amino acids of exon 10.
Whereas the former deletion translates into a severely truncated protein with loss of the proline-rich regions and the
WH2 domain,27 the inframe deletion of 52 amino acids results in a less truncated, stable protein of 450 amino acids,
demonstrable by Western blot, and contains all known functionally important domains of WASP, possibly explaining
the mild phenotype in JVT. A similar situation may account
for the mild WAS phenotype observed in a recently reported
family whose affected members had both Fanconi syndrome
and XLT. Sequence analysis of genomic DNA of these patients revealed a G insertion just one nucleotide downstream
of mutation in patient JVT.37
We conclude from this analysis that mutations affecting
the downstream portion of the WASP gene more effectively
interfere with the function of WASP, causing a severe phenotype. In contrast, missense mutations located within the PH
domain (exons 1 to 3) were consistently, with one exception,
associated with a mild phenotype, presumably by leaving
intact the functionally important cdc42 binding site, the SH3
binding motifs, and the WH2 domain, which includes a verprolin and cofilin homology domain.41 On the other hand,
genetic determinants not related to WASP and environmental
factors undoubtedly influence the clinical phenotype, possi-
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bly explaining the discordant WAS phenotypes observed in
members of some families.5,38
The proline-rich region of exon 10 contains multiple minimal PXXP motifs and two PPPPXXRG SH3 binding motifs
and is considered important for the interaction of WASP
with SH3-containing proteins.16,20-24 Using affinity precipitation and Western blot analysis, four of five SH3-GST fusion
proteins were found to bind to WASP in vitro, including
Fyn, Lck, PLC-g, and Grb2, suggesting that WASP may
selectively bind to many SH3-containing proteins. To explore whether mutated WASP loses its capability to bind to
SH3 domains, we analyzed WASP in cell extracts from BLCLs derived from selected patients with missense and nonsense mutations or splice-site mutations and deletions/insertions (not shown) by affinity precipitation of the Fyn-GST
fusion protein. Whenever the mutation allowed protein expression, the Fyn-GST fusion protein was able to bind the
mutated WASP, and the amount precipitated by Fyn SH3GST agarose beads correlated, in most instances, with the
amount of mutated WASP present in the cell lysate. Since
neither we nor others40 have yet found missense mutations
that selectively affect SH3 binding motifs, the importance
of these motifs for the function of WASP and the direct
interaction with SH3-containing proteins is unknown.
Although the function of WASP has not been clearly defined, recent observations support the hypothesis that WASP
plays a prominent role in the regulation of the actin/cytoskeleton system. Interaction of WASP with the small GTPase
cdc42, a key element in the dynamic organization of the
actin/cytoskeleton, has been suggested.25-27 However, missense mutations that would result in substitution of amino
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2688
ZHU ET AL
acids 238 to 257, the suggested cdc42 binding domain, have
not yet been identified. It is presently unknown whether
WASP interacts directly with actin or through actin binding
proteins, and if the underlying problem results in a transmembrane signaling defect. The observations that actin
bundling is necessary for T-cell activation by anti-CD3 antibody42 and that cdc42 is required for the polarization of
T cells toward antigen-presenting cells43,44 suggest that the
abnormal antibody responses characteristic of most WAS
patients are a direct consequence of defective T-/B-cell interaction. In the absence of functional WASP, T cells fail to
provide adequate help to B cells, resulting in impaired B-cell
function. Such a mechanism would explain the characteristic
defect in antibody responses to bacteriophage fX174 by
WAS patients,4,19 a pattern similar to that observed in patients
with X-linked hyper-IgM syndrome.45
ACKNOWLEDGMENT
The following individuals allowed us to study their patients and
provided material that was invaluable for this investigation: N. Day,
A. Filipovitch, C. Frantz, E. Gillian, R. Good, H. Hill, R. Insel, A.
Junker, A. Laszlo, J. Oleske, L. Pachman, R. Roberts, R. Schiff, A.
Shigeoka, C. Stotts, K. Sullivan, L. Vogler, and J. Winkelstein.
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1997 90: 2680-2689
Wiskott-Aldrich Syndrome/X-Linked Thrombocytopenia: WASP Gene
Mutations, Protein Expression, and Phenotype
Qili Zhu, Chiaki Watanabe, Ting Liu, Diane Hollenbaugh, R. Michael Blaese, Steven B. Kanner, Alejandro
Aruffo and Hans D. Ochs
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